Forgotten for two centuries: redescription of Phoxinus isetensis (Georgi, 1775) (Cypriniformes, Leuciscidae) – the most widespread minnow in Europe

Zoosyst. Evol. 100 (3) 2024, 1155-1173 | DOI 10.3897/zse.100.126702

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Forgotten for two centuries: redescription of Phoxinus isetensis
(Georgi, 1775) (Cypriniformes, Leuciscidae) — the most widespread
minnow in Europe

Oleg N. Artaev!, Aleksey A. Bolotovskiy', Ilya S. Turbanov!*°, Alexander A. Gandlin’?,
Aleksey V. Kutuzov', Marina A. Levina'*?, Danila A. Melentev*”, Ivan V. Pozdeev®,
Mikhail Ya. Borisov’, Boris A. Levin!*®

Papanin Institute for Biology of Inland Waters Russian Academy of Sciences, Borok, Russia
Severtsov Institute of Ecology and Evolution of the Russian Academy of Sciences, Moscow, Russia
Cherepovets State University, Cherepovets, Russia

Zoological Institute of the Russian Academy of Sciences, Saint Petersburg, Russia

Saint Petersburg University, Saint Petersburg, Russia

Saint Peterburg Scientific Center of the Russian Academy of Sciences, Saint Petersburg, Russia

NOD oO fF WY FE

Vologda branch of the Federal State Budget Scientific Institution "Russian Federal Research Institute of Fisheries and oceanography",
Vologda, Russia
8 Laboratory of Molecular Genetics, Russian Federal Research Institute of Fisheries and Oceanography, 105187 Moscow, Russia

https://zoobank. org/D0F 794A B6-6FEB-4F39-AA08-FA2A57B1B913

Corresponding authors: Oleg N. Artaev (artaev@gmail.com); Boris A. Levin (borislyovin@gmail.com)

Academic editor: Nicolas Hubert # Received 2 May 2024 # Accepted 15 July 2024 Published 22 August 2024

Abstract

The morphology, phylogenetic position, and distribution of a recently revalidated species of leuciscid minnow, Phoxinus isetensis,
were substantially clarified. The species was described in the late 18" century from the Middle Urals but later synonymized with
Phoxinus phoxinus. As believed, P. isetensis is distributed in the Arctic Ocean catchment from the Murman coast via West and East
Siberia until the Pacific Ocean catchment in Far East and Northeastern Asia. Our study, with the use of mtDNA markers coupled
with extensive morphological data, showed that the distribution of P. isetensis is greatly different. Currently, this is the most wide-
spread Phoxinus species in Europe, distributed in Northern and Western Europe and on the eastern edge of Siberia (Iset and Ural
basins). In particular, P. isetensis inhabits the basins of the Caspian, Baltic, White, Barents, and Kara seas, possibly occurring in the
North Sea basin. The species was redescribed, and the type locality and neotype were designated. The main morphological differ-
ence from other Phoxinus spp. is the large total number of vertebrae (39-43, mode 41) due to an increase in the number of caudal
vertebrae (16-21, mode 19). Phylogenetically, P. isetensis is a sister to the Caucasian species P. colchicus (p-distance = 5%). The
wide distribution of P. isetensis within the area of the Last Glacial Maximum suggests rapid colonization of deglaciated regions,
probably due to its adaptation to a cold climate.

Key Words

DNA barcoding, Europe-Siberia corridor, freshwater fish, postglacial expansion, taxonomy

Introduction family Leuciscidae Bonaparte, 1835, widespread in

northern Eurasia from the Pyrenees to the Pacific coast.
Minnows of the genus Phoxinus Rafinesque, 1820, are Initially, the morphology-based taxonomy of the genus
predominantly rheophilic small freshwater fish of the | Phoxinus was controversial due to the complex variability

Copyright Artaev, O.N. et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original author and source are credited.

1156

of morphological features and their large overlap. In early
comprehensive publications, all Phoxinus diversity was
reduced either to one species with a number of subspecies
(Berg 1949) or to several species (Kottelat and Freyhof
2007). The use of molecular genetic methods has shown
a greatly underestimated species diversity in Europe
(Palandaéic¢ et al. 2015, 2017, 2020), which suggested a
new look at the taxonomy of the genus Phoxinus. Only
two species (P. bigerri Kottelat, 2007 and P. colchicus
Berg, 1910) were supported by morphological and genetic
(mitochondrial) data, and six (P. phoxinus (Linnaeus,
1758), P. lumaireul (Schinz, 1840), P. karsticus Bianco &
De Bonis, 2015, P. septimanae Kottelat, 2007, P. marsilii
Heckel, 1836, and P. csikii Hanko, 1922) were supported
by mitochondrial but limitedly corroborated by nuclear
data (Palanda¢ic et al. 2017). Then several new species
were described using an integrative taxonomy approach:
P. krkae Bogutskaya, Jelic, Vuci¢, Jelic, Diripasko,
Stefanov & Klobuéar, 2019 (Bogutskaya et al. 2019),
P. dragarum Denys, Dettai, Persat, Daszkiewicz,
Hautecoeur & Keith, 2020, P. fayollarum Denys, Dettai,
Persat, Daszkiewicz, Hautecoeur & Keith, 2020 (Denys
et al. 2020), P abanticus Turan, Baycelebi, Ozulug,
Gaygusuz & Aksu, 2023 (Turan et al. 2023), P. radeki
Bay¢celebi, Aksu & Turan, 2024 (Bayc¢elebi et al. 2024),
and P. adagumicus Artaev, Turbanov, Bolotovskty,
Gandlin & Levin, 2024 (Artaev et al. 2024).

Along with the productive revision of the taxonomic
and genetic diversity of species distributed in Western
and Central Europe, the remaining part of the range, in-
cluding Eastern Europe, was almost unexplored. For in-
stance, taxonomic identification of northern and eastern
European minnows with the largest range in Europe was
problematic; those were assigned to mitochondrial Clade
17 without species naming (Palanda¢i¢ at al. 2017, 2020).
Our genetic data showed that unnamed Phoxinus sp.
Clade 17 is conspecific to recently revalidated Phoxinus
isetensis (Georgi, 1775) (Dyldin et al. 2023) described
from the Middle Urals. This study aimed to make tax-
onomic redescription using the integrative (morphology
and genetics) approach, to outline geographic distribu-
tion, and to clarify the phylogeny of P. isetensis.

Materials and methods
Sampling

Materials for morphological studies and partially for ge-
netic studies were collected by the authors. Fish were
caught using a frame net and seine net with a mesh size
of 6—8 mm. Fish were euthanized in a solution of clove
oil and photographed in an aquarium with artificial light-
ing using a Nikon D5300 camera (Nikon Corporation,
Tokyo, Japan) with a Nikkor 60 mm f/2.8G lens (Nikon
Corporation, Tokyo, Japan) using a physical white swatch
for color correction. Fin clips (pectoral or pelvic) were
taken from some specimens (DNA vouchers) and placed

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Artaev, O.N. et al.: Redescription of Phoxinus isetensis — the most widespread minnow in Europe

in 96% ethanol for subsequent DNA extraction in the
laboratory. Then most fish were preserved in 10% for-
malin (form.), while some samples (usually small-sized
specimens) were preserved in 96% ethanol for molecular
analysis. Subsequently, formalin-fixed specimens were
washed out in running water and transferred to 70% etha-
nol for long-term storage.

Neotype and additional material were deposited at the
Fish Collection of the Papanin Institute for Biology of In-
land Waters of the Russian Academy of Sciences, Borok,
Russia (IBIW_ FS).

Morphological studies

Morphological material on Phoxinus sp. (Clade 17) from
seventeen localities (n=272, Fig. 1, Suppl. material 1) was
examined. In studying the morphology of Phoxinus, we
follow Bogutskaya et al. (2019, 2023) and Artaev et al.
(2024). In particular, 42 morphometric (Suppl. materials
2, 3), 17 meristic, and two qualitative characters (Suppl.
materials 2, 4) were processed. Abbreviations of morpho-
metric characters are seen in Suppl. material 3. Morpho-
metric measurements were taken from the left side of the
body using a digital caliper to the nearest 0.1 mm by one
operator for the purposes of consistency as recommended
by Mina et al. (2005). Meristics (except for axial skeleton)
and type of breast scalation (Bogutskaya et al. 2019) were
assessed using material stained in an ethanol solution of
alizarin red S (Taylor and Van Dyke 1985 with modifi-
cations), followed by short exposure to 1—2% potassium
hydroxide and preservation in 70% ethanol.

Sex was determined by the shape and size of the pecto-
ral fins, their rays, and the length of the pelvic fins (Frost
1943; Berg 1949; Chen 1996; Bogutskaya et al. 2019).
External meristics were counted on the left side. Stan-
dard length (SL) was measured from the tip of the upper
lip to the end of the hypural complex. The total number
of pectoral and pelvic-fin rays was counted on the left
fins. The last two branched rays articulated on a single
pterygiophore in the dorsal and anal fins are counted as
one. Scales above the lateral line were counted between
the lateral line and base of the first unbranched ray in the
dorsal fin; scales below the lateral line were between the
lateral line and base of the first unbranched ray in the anal
fin. In both cases, lateral line scales were not taken into
account. The number of anterior gill rakers of the first
gill arch was counted on the left and right sides of the
specimens. Number lines on the scales were counted on
the left and right breast patches, and an average value was
taken. The counts of meristic characters (except for the
axial skeleton) and assessment of qualitative characters
were done using the stereomicroscope MC-2-ZOOM
(Micromed, Saint Petersburg, Russia). Vertebrae and
pterygiophores were counted following Naseka (1996)
and Bogutskaya et al. (2019) using radiographs made by
X-ray equipment PRDU IIT (ELTECH-Med, Saint Peters-
burg, Russia). Images of pharyngeal teeth were obtained

Zoosyst. Evol. 100 (3) 2024, 1155-1173

30°W

20°W 10°W 0° 10°E 20°E

10°E 20°E 30°E

30°E 40°E 50°E 60°E 70°E 80°E 90°E

40°E 50°E 60°E

Figure 1. Distribution map and range boundaries of Phoxinus isetensis confirmed by morphological and genetic data along with other
confirmed Phoxinus spp., whose distributions are partially within the glacier-covered area of the last glacial maximum. Locality num-

bers are designated in Suppl. materials 1, 5. *

using a JEOL JSM-6510LV scanning electron micro-
scope (Jeol, Tokyo, Japan).

Measurement indexes were statistically processed in
Microsoft Excel. Comparison of multiple samples was
carried out using the Kruskal-Wallis test followed by
Dunn’s post hoc test with Bonferroni correction [rstatix
(Kassambara 2020) and tidyverse (Wickham et al. 2019)
packages in R version 4.3.1 (Ihaka and Gentleman 1996)].
Principal component analysis (PCA) was performed us-
ing the ggfortify (Tang et al. 2016) package in R. Differ-
ences between sexes were tested using the Mann-Whit-
ney U test in Past 4.13 (Hammer and Harper 2001).

Phylogenetic placement and genetic distance.

DNA was isolated by salt extraction (Aljanabi and Marti-
nez 1997) from ethanol-fixed tissues. Two mitochondrial
markers were analyzed. The mitochondrial cytochrome
c oxidase subunit I (COI) barcode region was amplified
using the M13-tailed primer cocktail: FishF2_ tl: 5'-TGT
AAA ACG ACG GCC AGT CGA CTA ATC ATA AAG
ATA TCG GCA C-3', FishR2_ tl: 5'-CAG GAA ACA GCT
ATG ACA CTT CAG GGT GAC CGA AGA ATC AGA
A-3', VF2_tl: 5'-TGT AAA ACG ACG GCC AGT CAA

— by Palandaéti¢ et al. (2020); **

— by Palandaéi¢ et al. (2020) and Rothe et al. (2019).

CCA ACC ACA AAG ACA TTG GCA C-3', and FR1d_
tl: 5'-CAG GAA ACA GCT ATG ACA CCT CAG GGT
GTC CGA ARA AYC ARA A-3' (Ivanova et al. 2007).
PCR conditions for COI followed protocols from Ivanova
et al. (2007). In addition, the cytochrome 5 (cytb) frag-
ment was amplified by PCR using the following primers:
GluF: 5'-AACCACCGTTGTATTCAACTACAA-3' and
ThrR: 5'-ACCTCCGATCTTCGGATTACAAGACCG-3'
(Machordom and Doadrio 2001). PCR amplifications
were performed using Evrogen ScreenMix-HS under
conditions described by Levin et al. (2017).

Sequencing of the PCR products, purified by ethanol and
ammonium acetate (3 M) precipitation, was conducted us-
ing the Applied Biosystems 3500 DNA sequencer (Thermo
Fisher Scientific, USA), with primers M13F 5'-GTA AAA
CGA CGG CCA GT-3' M13R-pUC 5'-CAG GAA ACA
GCT ATG AC-3' (Geiger et al. 2014) for COI and primers
GluF: 5'-AACCACCGTTGTATTCAACTACAA-3' and
ThrR: 5'-ACCTCCGATCTTCGGATTACAAGACCG-3'
(Machordom and Doadrio 2001) for cytb.

DNA chromatograms were checked for errors in
FinchTV 1.4.0 (Rothganger et al. 2006), and the DNA
sequences were aligned using the ClustalW algorithm
in MEGA7 (Kumar et al. 2016). Phylogenetic analy-
sis was performed on COI (567 bp) and cytb (1089 bp)

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1158

concatenated sequences. In addition to the 29 newly de-
termined COI and cytb sequences in this study, 294 con-
catenated sequences of all available Phoxinus spp. were
mined from the GenBank (derived from the studies of
Imoto et al. 2013; Xu et al. 2014; Palanda€ic et al. 2015,
2017, 2020; Ramler et al. 2016; Xie et al. 2016; Schon-
huth et al. 2018; and unpublished works). Three out-
groups representing the genera Pseudaspius Dybowsk1,
1869, Rhynchocypris Gunther, 1889, and Oreoleuciscus
Warpachowski, 1889, were selected according to the pre-
vious phylogenetic studies (Palanda¢éi¢ et al. 2015, 2020)
(Suppl. material 5). Only unique haplotypes were used in
downstream phylogenetic analyses.

The Bayesian phylogenetic analysis was performed in
a Bayesian statistical framework implemented in BEAST
v.1.10.4. (Hill and Baele 2019) with 2x10’ MCMC gen-
erations (10% burn-in) and parameters sampled every
2000 steps. The substitution models by codon position for
Bayesian analysis were selected in PartitionFinder v.2.1.1
(Lanfear et al. 2016) with the greedy algorithm (Lanfear
et al. 2012) (Suppl. material 6).

Maximum likelihood phylogenies were inferred us-
ing IQ-TREE v.2.2.0 (Nguyen et al. 2015) in PhyloSuite
v.1.2.3 (Zhang et al. 2020; Xiang et al. 2023) under the
edgelinked partition model for 1000 ultrafast (Minh et
al. 2013) bootstrapping. ModelFinder v.2.2.0 (Kalyaana-
moorthy et al. 2017) in PhyloSuite v.1.2.3 was used to
select the best-fit partition model (edge-linked) using the
AICc criterion (Suppl. material 6).

The average intra-group as well as the average pairwise
intergroup p-distances using the concatenated COI+cytb
sequences data set were calculated using the MEGA7 pro-
gram (Kumar et al. 2016) with 1000 bootstrap replicas.

Map visualization

The map was created using the QGIS software, v.3.34. Dig-
ital elevation model visualized based on GMTED2010, 30
sec. resolution (Danielson and Gesch 2011); river systems
— HydroATLAS v.1.0 (Linke et al. 2019); LGM ice sheet
boundary according to Batchelor et al. (2019).

Results

Phylogenetic placement and genetic distance

The phylogenetic Bayesian tree of the genus Phoxinus
shows that P. isetensis has its own lineage, being sis-
ter to P. colchicus distributed in the eastern Black Sea
basin and the Kuban system in the Sea of Azov basin
(p-distance = 0.050+0.005) with a high support in both
BI (Fig. 2) and ML (Suppl. material 7) analyses. Three
species combined together (P. isetensis, P. colchicus, and
P. chrysoprasius) are early branching in the Phoxinus tree
and represent a sister class to all other European Phox-
inus spp. apart from P. adagumicus, although this was

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Artaev, O.N. et al.: Redescription of Phoxinus isetensis — the most widespread minnow in Europe

weakly supported. Intraspecies divergence of P. isetensis
is moderate (0.005) despite its wide distributional range
(Suppl. material 8).

Systematics

Class Actinopterygii Klein, 1885
Order Cypriniformes Bleeker, 1859
Family Leuciscidae Bonaparte, 1835
Genus Phoxinus Rafinesque, 1820

Phoxinus isetensis (Georgi, 1775)
Figs 3, 4

English name: Northern Minnow; Russian name: CepepHblit roJIbaH

Cyprinus phoxinus — Linnaeus 1758: 322 (Europa (part)); Falk 1786:
432 (Volga, Tsaritsa, Elshanka, Sarpa, etc.); Fischer 1791: 258 (Li-
vonia); Hupel 1777: 467 (Liffland and Estonia).

Cyprinus aphya — Linnaeus 1758: 323 (European rivers (part)); Fischer
1791: 258 (Livonia); Georgi 1775: 881 (Sukhona River); Falk 1786:
429 (Kama R. and its tributaries).

Cyprinus (without Latin species name) — Lepechin 1771: 491 (circa
Catharinopolin).

“Taian” or “cougars” (without Latin species name) — Lepechin 1772:
309 (upstreams of the Isset, Chusovaya, and Tura rivers).

Cyprinus, “Krasnosobik” or “Soldat” (without Latin species name)—
Georgi 1775: 550 (Iset River).

Cyprinus isetensis Georgi 1775: 621 (Chusovaya River).

Cyprinus galian Gmelin 1789: 1421 (vicinities of Yekaterinburg).

Phoxinus rivularis — Watecki 1864: 50 (Neman River).

Phoxinus laevis — Kessler 1864: 124 (Neva River); Kessler 1870: 268
(Volga, Samara basins, Khmelevka creek near Vasilsursk); War-
pachowski 1889: 61 (Volga R. system in Nizhny Novgorod prov-
ince); Sabaneev 1892: 423 (Yaroslavl and Perm province, near Mos-
cow: rivers Lichoborka and Sinichka (trib. of Jausa R.), Moskva R.
at Kamenny most); Dybowski 1862: 105 (Livonia).

Tinca phoxinus — Plater 1861: 37, 63 (Daugava River).

Phoxinus phoxinus — Berg 1912: 260 (Finland, Kola region, Europe-
an rivers of the Arctic Ocean basin); Berg 1923: 166 (in Russia all
over); Berg 1932: 368 (in Arctic Ocean basin from Murmansk east-
ward, Volga basin upstream Syzran (include Kama and Oka rivers),
possible in Ural River); Berg 1949: 588 (same place); Reshetnikov
et al. 2003: 301 (widespread in Europe (part)); Kottelat and Freyhof
2007: 228 (Scandinavia and Russia’s northernmost extremity; Up-
per and middle Volga, Ural).

Phoxinus isetensis — Dyldin et al. 2023: 36 (Arctic Ocean basin, from
Murman coast to East Siberian Sea basin (part)).

Phoxinus sp. — Dyldin et al. 2023: 37 (Europe, in North Sea, Baltic Sea
basins (including Gulf of Finland and Neva River), and northern
Caspian Sea basin (Upper Volga River, including Kama and Oka
rivers, probably in Ural River)).

Type material. Neotype, female (SL 63.9 mm, IBIW_
FS_422, Genbank Accession numbers PP538745—COI,
PP548200-cytb), Russia, Sverdlovsk Region, Ob River
basin, Severka River (Tobol River basin) upstream Sever-
ka village near Yekaterinburg, 56.8830°N, 60.2716°E,

Zoosyst. Evol. 100 (3) 2024, 1155-1173

93/*

1159

Pseudaspius hakonensis
Rhynchocypris lagowskii
Oreoleuciscus potanini

“l P. adagumicus, Kuban basin
P. umonensis, Irtysh basin

P. sp., Amur basin

P. phoxinus tumensis,
Sea of Japan basin

*/%

P. chrysoprasius, Salgir basin
-l- 7 P. colchicus, Black Sea basin,

*
cel
*

87/-
of
a
aie
98/0.96

99/0.83

95/0.94 ------+

0.03

af*

sd ha P. isetensis, Baltic, Barents, Caspian,
Kara, White Seas basins

P. cf. morella, North Sea basin

*/*7| P. septimaniae, Adriatic, North,
Mediterranean Seas basins

*/*/) P. phoxinus, North Sea basin,
Danube basin

P. marsilii, Danube, Vistula, Oder basins
{ P. sp. (Clade 8), Adriatic Sea basin
wi “<| P. karsticus, Adriatic Sea basin
{ P. krkae, Adriatic Sea basin

cl P. csikii (Clade 5a), Danube basin
SS P. csikii (Clade 5b), Danube basin,

Mediterranean Sea basin
wa P. sp. (Clade 4), Danube basin

P. sp. (Clade 3), Danube basin

-/0.83

P. sp. (Clade 2), Danube basin

ae P. sp. (Clade 2), Adriatic Sea basin,
Danube basin

P. strandjae, Black Sea basin

P. strymonicus, Strymon basin

P. lumaireul (Clade 1e), Danube basin

x P. lumaireul (Clade 1f), Danube basin

<

P. lumaireul (Clade 1d), Danube basin

P. lumaireul (Clade 1b), Danube basin

P. lumaireul (Clades 1a, 1b, 1c),
Adriatic Sea basin, Danube basin

HO.18 | P. lumaireul (Clade 1b), Danube basin
P. lumaireul (Clade 1b), Adriatic Sea
basin, Danube basin

P. lumaireul (Clade 1b), Danube basin

P. lumaireul (Clade 1a), Adriatic Sea
basin

P. lumaireul (Clade 1a), Adriatic Sea
basin, Danube basin

97/0.99

Figure 2. BI consensus tree of concatenated COI and cythb mtDNA sequences representing available Phoxinus species in the NCBI
data base combined with our data set. The numbers of some yet-unnamed clades are given according to the study of Palandacic¢ et
al. (2020). Phoxinus isetensis 1s highlighted with color. Bootstrap values/posterior probabilities above 70/0.7 are shown; asterisks
represent 100/1 bootstrap/posterior probabilities values. The scale bar represents the expected substitutions per site. The nodes with
multiple specimens were collapsed into a triangle, with the horizontal depth indicating the level of divergence within the node.

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1160

Artaev, O.N. et al.: Redescription of Phoxinus isetensis — the most widespread minnow in Europe

ae
i

ta tae ala AT

Figure 3. Neotype of Phoxinus isetensis (SL 63.9 mm, IBIW_FS_422, female). A. Live appearance; B. General appearance of the

preserved specimen; C. Radiograph.

21 June 2023, O.N. Artaev, I.S. Turbanov, A.A. Bolo-
tovskiy leg.
Additional material. see Suppl. material 1.
Comparative material. see Suppl. material 1.
Etymology. Since Lepechin (1771: 491) described spe-
cies from the vicinity of Yekaterinburg, it can be assumed
that Georgi (1775: 621) (see taxonomic remarks) gave its
name to the Iset River, flowing through Yekaterinburg.
Diagnosis. Phoxinus isetensis is distinguished from
other European minnows (P. adagumicus, P. chrysopra-
sius, P. colchicus, P. csikii, P. krkae, P. lumaireul (Clade
laand Clade 1b), P. marsilii, P. septimaniae, P. strandjae,
and Phoxinus sp. (Clade 2) by having a number of total
vertebrae (39-43, mean 41.0, mode 41) and a number of
caudal vertebrae (16-21, mean 18.9, mode 19).
Phoxinus isetensis is further distinguished from min-
nows from Eastern Europe (P. adagumicus, P. chryso-
prasius, and P. colchicus) by a longer caudal peduncle
(caudal peduncle length 2.5—3.7, mean 3.1 times caudal
peduncle depth); fewer circumpeduncular scales (28-45,
mean 35.3); fewer scale rows above the lateral line (10-
21, mean 15.1); and a combination of characters, none

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of which is unique, as follows: eye horizontal diame-
ter 5.9-8.8% SL, mean 7.2 and eye horizontal diameter
23.4-33.8% HL, mean 28.1; depth of caudal peduncle
6.6—-9.0% SL, mean 7.7 in females and 7.2—9.5, mean 8.2
in males; caudal peduncle length 20.5—26.9% SL, mean
23.9 in females and 22.9—-27.7 mean 25.1 in males; 8—16
scale rows below lateral line (mean 11.2, mode 11) (Sup-
pl. materials 3, 4).

Description. The live and preserved appearance as well
as radiograph of neotype is shown on Fig. 3, general ap-
pearance of live specimens of Phoxinus isetensis from dif-
ferent basins 1s shown on Fig. 4, morphometrics of neotype
and additional material from the type locality with level of
significance of sex-related differences are given in Table 1,
meristic and qualitative characters for specimens from the
type locality are given in Table 2, and primary morpho-
logical data for specimens from the type locality (neotype
and additional material) are given in Suppl. material 2, the
meristic and qualitative characters of P. isetensis and Phox-
inus spp. are given in Suppl. material 4, the morphometrics
of P. isetensis, P. adagumicus, P. chrysoprasius, and P. col-
chicus and their comparison are given in Suppl. material 3.

Zoosyst. Evol. 100 (3) 2024, 1155-1173 1161

Figure 4. Live appearance of Phoxinus isetensis from different basins. A. Male in pre-spawning coloration, SL 48.5 mm, Nataleyka
R. (Middle Volga basin), 53.9781°N, 45.6530°E, 05 May 2022; B. Female in pre-spawning coloration, SL 48.8 mm, same location
and date; C. Female, SL 64.3 mm, Okhomlya R. (Baltic Sea basin), 58.7078°N, 33.5199°E, 12 September 2021; D. Female, SL 62.4
mm, Kyltymyu River (Northern Dvina basin), 61.4981°N, 50.5831°E, 21 September 2022; E. Male, SL 59.5 mm, Marat Bai River
(Ural basin), 54.0668°N, 58.8038°E, 4 June 2023; F. Female, SL 63.6 mm, Karnasyavryok River (Barents Sea basin), 68.9314°N,
34.9318°E, 27 May 2023.

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1162 Artaev, O.N. et al.: Redescription of Phoxinus isetensis — the most widespread minnow in Europe

Table 1. Morphometrics of Phoxinus isetensis from the type locality (Severka River) (mean+SD - bold, and ranges - narrow) with
level of significance of sex-related differences (primary data see in Suppl. material 2). * Difference between females and males,
Mann-Whitney U test: ns (p > 0.05), + (p < 0.05), +4 (p < 0.01).

Morphometric characters Neotype (female) females, n=11 = males, n=9 p*
SL 63.9 56.5+3.7 50.5+2.7
50.9-63.9 46.2-54.9
In percentage of standard length (% SL)
Body depth at dorsalfin origin 18.0 18.3+1.4 18.7+1.0 ns
15.9-20.2 16.7-20.0
Body width at dorsal-fin origin 12.6 13.4+0.8 12.6+1.2 ns
11.8-14.4 11.1-14.8
Minimum depth of caudal peduncle 6.9 7.7+0.4 8.3+0.4 ns
6.9-8.3 7./-8.8
Caudal peduncle width 8.7 9.0+0.4 9.1+0.6 +
8.3-9.6 7.5-9.7
Predorsal length 56.2 56.5:+0.7 55.6+1.3 +
55.5-58.3 54.3-58.7
Postdorsal length 34.2 33.8+1.3 34.2+0.9 ns
32.1-36.2 32.7-35.7
Prepelvic length 47.8 48.6+1.2 47.5+0.8 ns
47.1-51.1 46.1-48.4
Preanal length 64.6 65.0+1.5 63.4+0.9 ns
62.1-67.7 62.0-65.2
Pectoral — pelvic-fin origin length 23.6 24.8+1.2 23.0+1.1 ++
23.2-26.7 21.3-25.0
Pelvic — anal-fin origin length LA 17.8+0.7 17.6+0.8 ns
16.5-18.9 16.6-19.0
Caudal peduncle length 24.1 23.9+1.0 25.3+0.8 ns
22.0-25.1 24.1-26.4
Dorsalfin base length 11.0 11.1+0.6 11.6+0.6 ns
10.4-12.5 10.7-12.5
Dorsalfin depth 18.6 18.9+0.7 20.8+1.0 +
17.8-20.1 18.9-21.9
Anal-fin base length 11.4 10.6+0.5 10.8+0.6 ns
9.7-11.4 10.0-11.6
Anal-fin depth 18.6 18.7+0.7 19.8+0.8 ++
17.5-19.9 18.7-21.1
Pectoralfin length Lt 17.6+1.1 19.8+0.9 -
16.3-20.1 18.1-20.7
Pelvic-fin length 132 13.7+0.7 15.8+0.8 ~
12.9-15.4 14.7-17.1
Head length 25.6 25.2+0.5 26.0+0.7 ns
24.4-25.8 24.8-27.5
Head depth at nape 155 15.5+0.5 16.4+0.4 ns
14.4-16.4 15.7-16.9
Maximum head width 13.9 13.6+0.5 13.6+0.6 ns
12.7-14.4 12.4-14.5
Snout length Fe 7.6+0.3 7.8+0.3 ns
7.2-8.1 7.5-8.4
Eye horizontal diameter one! 6.9+0.3 7.1+0.3 +
6.5-7.4 6.6-7.6
Interorbital width 8.5 8.8+0.5 8.7+0.6 ns
8.2-9.7 7.6-9.6
In percentage of head length (% HL)
Maximum head width 54.3 54.0+1.6 52.5+2.8 ns
51.5-56.0 47.7-55.9
Snout length 28.4 30.1+1.1 30.1+1.3 ns
28.4-31.6 28.3-32.6
Head depth at nape 60.4 61.3+1.8 63.2+1.8 ns
58.6-64.4 60.6-65.3
Head depth through eye 48.3 48.7+1.0 49.9+1.5 ns
47.4-50.8 48.1-52.2
Eye horizontal diameter 26.2 27.4+0.8 27.4+1.3 ++
26.2-28.8 25.3-29.6

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Zoosyst. Evol. 100 (3) 2024, 1155-1173

Morphometric characters
Postorbital distance

Interorbital width

In percentage of caudal peduncle length
Minimum depth of caudal peduncle

In percentage of body depth
Head length

In percentage of interorbital width
Eye horizontal diameter

Ratios:
Interorbital width/eye horizontal diameter

Snout length/eye horizontal diameter

Head depth at nape/eye horizontal diameter

Head length/caudal peduncle depth

Length of caudal peduncle/caudal peduncle depth
Pectoral fin length/pectoral — pelvic-fin origin distance
Predorsal length/head length

Body width at dorsal-fin origin/Caudal peduncle depth

Morphometrics (Table 1, Suppl. material 3). The max-
imum size among studied specimens 76.3 mm SL. The
species has a slender and elongated caudal peduncle. The
caudal peduncle depth 6.9% SL in neotype, 6.9-8.8% SL
in additional material from type locality, and 6.6-9.5%
SL in other additional materials (here and further — from
basins of Caspian, Baltic, Barents, and Kara seas): cau-
dal peduncle depth 28.7% in caudal peduncle length in
neotype, 28.7—36.5 in additional material from type lo-
cality, and 26.9-40.6 in other additional material; caudal
peduncle depth 3.5 times the caudal peduncle length in
neotype, 2.9—3.4 in additional material from type locali-
ty, and 2.5—3.7 in other additional material. The species
has a slender body, body depth at dorsal-fin origin 18.0%
SL in neotype, 15.9-20.0 in additional material from
type locality, and 14.7—21.6 in other additional material.
Eyes larger (horizontal eye diameter: 26.2% HL in neo-
type, 25.3—29.6 in additional materials from type locality,
23.4—33.8 in other additional).

Meristics (Table 2, Suppl. material 4). Dorsal fin with 3
(very rarely 2) unbranched and (8) 7/2 (6) branched rays.
Anal fin with 3 unbranched and (6) 72 (8) branched rays.
Pectoral fin with 14-20, commonly 16-18 rays. Pelvic
fin with (7) 8 (9) rays. Caudal fin with (18) 19 (20) rays.

Among 135 individuals, the most common pharynge-
al teeth formula is classic for the genus 2.5-4.2 (n=102)
(Fig. 5A; Suppl. material 4). Other variants are 2.54.1
(n=10), 2.44.2 (n=8), 1.5—-4.2 (n=4), 2.5—5.2 (n=3), 1.5-
4.1 (n=2), 2.44.1 (n=2), 2.3-4.2 (n=1), 2.5-4.3 (n=1),

1163

Neotype (female) females, n=11 = males, n=9 p*
45.6 44.5+1.1 44.8+2.1 +
42.4-45.9 41.9-47.6
Sot 34.9+2.0 33.6+1.9 ns
31.7-38.6 29.5-35.4
28.7 32.4+2.0 32.8+2.2 ns
28.7-35.0 29.4-36.5
142.5 138.3+10.6 139.5+8.9 +
127.1-159.6 129.6-155.2
78.4 79.0+5.2 81.8+4.1 +
72.4-91.0 75.5-85.8
T3 1.3+0.1 1.2+0.1 +
1.1-1.4 1.2-1.3
ied 1.1+0.0 1.1+0.1 ns
1.0-1.2 1.0-1.2
23 2.2+0.1 2.3+0.1 ++
2.0-2.4 2.2-2.4
37 3.320.2 3.1+0.2 +
3.1-3.7 2.9-3.4
329) 3.120.2 3.1+0.2 ns
2.9-3.5 2./-3.4
0.7 0.7+0.1 0.9+0.1 ++
0.7-0.9 0.7-0.9
Zee 2.2+0.0 2.1+0.1 ++
2.2-2.3 2.0-2.3
1.8 1.7+0.1 1.5+0.1 +
1.6-1.8 1.4-1.7

2.3.5-4.3.2 (n=1). Among those one is exceptionally rare
for phoxinin fishes — three-rowed formula 2.3.5-4.3.2
that was recorded in an individual from the Tsna R., Bal-
tic Sea basin.

Forty-two total vertebrae in neotype, 40-42 in addi-
tional material from type locality, and 39-43 in other ad-
ditional material from basins of Baltic and Barents seas,
Volga and Ob rivers, commonly 41 vertebrae. Twenty-one
abdominal vertebrae in neotype, 21—23 in additional ma-
terial from type locality, and 21—24 in other additional
material from basins of Baltic and Barents seas, Volga
and Ob rivers, commonly 21—23 vertebrae. Twenty-one
caudal vertebrae in neotype, 17—21 in additional material
from type locality, and 16—21 in other additional material
from basins of Baltic and Barents seas, Volga and Ob riv-
ers, commonly 18—20 vertebrae. Fourteen predorsal ver-
tebrae in neotype, 14-16 in additional material from type
locality, and 13—16 for other additional material from ba-
sins of Baltic and Barents seas, Volga and Ob rivers, com-
monly 14—15 vertebrae. Three anal-fin pterygiophores in
front of the first caudal vertebrae in neotype, 3—6 in ad-
ditional material from type locality, and 3—7 in other ad-
ditional material from basins of Baltic and Barents seas,
Volga and Ob rivers, commonly 4—6 pterygiophores. Dif-
ference between numbers of abdominal and caudal verte-
brae zero in neotype, zero to 6 in additional material from
type locality, and zero to 7 for other additional material
from basins of Baltic and Barents seas, Volga and Ob riv-
ers, commonly 2-5.

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1164

Table 2. Meristic characters of Phoxinus isetensis from the type
locality (Severka River) (primary data see in Suppl. material 2).

MeansSD_ in
(or mode)
range
86.124.4 10
80-93
50.8215.1 10
23-66
Number of pored scales in first complete (non- 20.8412.7 10

Characters

Total number of scales in lateral series (Sql)

Total number of lateralline (pored) scales (It)

interrupted) section of lateral line (Ilcs) 1-45
Relative number of total lateral-line scales, 0.60+0.19 10
quotient Ilt: sql (Iltr) 0.26-0.83
Mean number of scale rows on left and right 8.4+1.0 8
breast patches (BrPScale) 7.5-10.5
Number of circumpeduncular scales (cps) 35.2+1.6 10
33-39
Scales above lateral line (between lateral line 15.4+1.3 10
and base of first unbranched ray in D) (all) 14-18
Scales below lateral line (between lateral line 11.4+1.6 10
and base of first unbranched ray in A) (bll) 9-14
Pattern of scalation on the breast and anterior 4 4-6 8
belly (cstyp)
Total number of pectoralfin rays (P)left 16.4+0.8 10
15-18
Total number of pelvic-fin rays (V) 8.020.5 10
7-9
Number of branched dorsalfin rays (D) 7.0+0.0 10
7-7
Number of branched anal-fin rays) (A) 7.00.0 10
7-7
Number of rays in caudal fin (C) 18.7+0.5 10
18-19
Total number of vertebrae (tv) 40.9+0.6 30
40-42
Number of abdominal vertebrae (abdv) 21.9+0.7 30
21-23
Number of caudal vertebrae (caudv) 19.0+0.9 30
17-21
Number of predorsal abdominal vertebrae 14.7+0.5 30
(preDv) 14-16
Number of analfin pterygiophores in front of 4.9+0.9 30
the first caudal vertebrae (preAp) 3-6
Difference between numbers of abdominal 2.9+1.5 30
and caudal vertebrae (dac) 0-6
Gill rakers in first arch 8.5+0.8 10
7-10

Seventy-one to 103 (mean 85.7) total number of scales
in the lateral series. Lateral line incomplete and interrupt-
ed. The relative number of total lateral-line (pored) scales
varies greatly from 12% to 99%, mean 61%. Five to 11
(commonly 6—9) scale rows on breast patches. 28-45
(mean 35.2) circumpeduncular scales. Ten to 21 (mean
15.1) scale rows above lateral line. Eight to 14 (16), mean
11.2 scale rows below lateral line.

Seven to 10 (mode 8) gill rakers (in series from type
locality) on first arch.

Qualitative characters. Pectoral fins do not reach the
beginning of pelvic fins in females and most of the males
(ca. 75%). In the most specimens (ca. 85%) tip of the upper
lip above horizontal level of lowest point of the eye and in

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Artaev, O.N. et al.: Redescription of Phoxinus isetensis — the most widespread minnow in Europe

about 5% of specimens — at this level. Origin of anal fin
is mainly ahead or at vertical level of posterior insertion
of the dorsal fin (ca. 45% for each form), rarely behind
(ca. 8%). Free margin of the dorsal fin 1s mainly straight or
slightly convex, rarely slightly concave; anal fin most often
slightly concave and straight, rarely slightly convex. 3-6"
type of breast scalation (mode 4" type, often 3" type, 5"
and 6" type are less common) (Fig. 5B, Suppl. material 4).

Coloration. Males and females outside of spawning
have predominantly brown coloring of the upper half of
the body and light lower part in males and white in females
(Fig. 4). Juveniles often show a large contrast: the black
horizontal stripe and the white belly. During spawning,
color of both sexes becomes much brighter, the color of
the sides is dominated by green (many males become dark
green, almost black), in front, it is mixed with golden, less
often purple and red. In males, as well as some females, the
lips and lower jaw, as well as body at the bases of the pec-
toral, pelvic, and anal fins, become red. The operculum is
blue and the suboperculum is yellow in both sexes, but col-
oration is much more pronounced in males. In both sexes,
the bases of ventral and anal fins are light blue. The spec-
imens preserved in formalin had a yellowish color, which
is somewhat darker with a brown tint in the upper parts.

Sexual dimorphism. Significant differences are ob-
served in 18 out of 41 morphometric characters (Table
1). In general, females have smaller relative anal (anal-
fin depth), dorsal (dorsal-fin depth), pelvic (pelvic-fin
length), and pectoral fins (pectoral-fin length), a greater
predorsal length, and pectoral — pelvic-fin origin length.
In females, the pectoral fins never reach the pelvic fins,
while in ca. 25% of males, reach.

Taxonomic remarks. According to the early literary
sources reviewed in Berg (1912), the first name within the
range of species 1s Cyprinus isetensis, given by Georgi
(1775: 621), which lists species (without description) for
the Chusovaya River with reference to Lepechin (1771:
491). Lepechin gives a description of the species but does
not give the species name, designated as “CYPRINVS”
with the type locality “circa Catharinopolin” (now Yekat-
erinburg). Although Georgi does not provide a description
of species when mentioning the name isetensis, he makes
a reference to the description of this species in Lepechin’s
study, which makes this name valid since it complies with
Art. 12.1 and 12.2 of the International Code of Zoological
Nomenclature (ICZN 1999).

Type locality. The type locality from the original
description (Lepechin 1771: 493) is “... habitat in rivis
scopulosus circa Catharinopolin,” which means “... lives
in the rocky streams around Catharinopolin (now Yekat-
erinburg).” Probably Lepechin meant the upper reaches
of the Iset, Chusovaya, and Tura rivers, which were spec-
ified in further publication (Lepechin 1772: 311).

Type locality for the neotype: Severka River
(56.8830°N, 60.2716°E) upstream of Severka village
near Yekaterinburg, Sverdlovsk Oblast, Russia (Fig. 6).
A tributary of the Reshotka River — Iset River — Tobol
River — Irtysh River — Ob River — Kara Sea.

Zoosyst. Evol. 100 (3) 2024, 1155-1173

1165

Figure 5. Morphological features of Phoxinus isetensis. A. Most frequent variant of the formula of pharyngeal bones: double-rowed
formula 2.5—4.2, scale bar 0.5 mm; B. Ventral view of alizarin-stained female and male from the Bekshanka River (Volga basin).
Female had 5" type scalation on breast and belly, male had 6" type.

Nomenclatural and taxonomic actions. The need to
designate a neotype for P. isetensis is determined by the
following considerations: first, our attempts to find a type
specimen at the Zoological Institute of the Russian Acad-
emy of Sciences, Saint Petersburg, Russia (ZISP), where
the largest and oldest ichthyological collection in Russia
is stored, were unsuccessful. At the end of the 18" centu-

ry, at the time of the description of P. isetensis, it was the
only scientific organization in Russia where type speci-
mens were deposited. The type specimens of P. isetensis
were absent in the ZISP already at the beginning of the
20" century (Berg 1912). Second, no type specimens for
all the fish species described by the naturalist and explor-
er Johann Gottlieb Georgi (1729-1802) were designated

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1166

(Fricke et al. 2024). Thus, we conclude that Georgi did
not designate a type series for this species. Third, in addi-
tion to P. isetensis, at least one more species of this genus
inhabits the Ob River basin—P. ujmonensis Kashchen-
ko, 1899 (see Discussion), and their possible sympatric
co-occurrence requires further clarification.

Thus, based on the above-mentioned circumstances
and in accordance with Article 75 of the ICZN, we desig-
nate a neotype for P. isetensis. Our nomenclatural actions
do not contradict the statements of Article 75.3 (qualifying
conditions), and the designation of a nomenclatural type
(neotype) for P. isetensis, as a widespread species living in
Europe and Asia, will make it possible to clearly describe
both morphological and genetic differences from other
species of the genus Phoxinus (Articles 75.3.1 and 75.3.2).

Distribution and habitat. Widespread in northern and
eastern Europe and in the western edge of Siberia (Iset
and partially Ural basin). Phoxinus isetensis inhabits the
basins of the Caspian, Baltic, White, Barents, and Kara
seas, possibly occurring in the North Sea basin. In the
Caspian Sea basin, it is widely distributed in the upper
and middle Volga, Kama, in the mountainous part of the
Ural basin. In the Baltic Sea basin, it is widespread in
the northern and eastern parts. In the Kara Sea basin, it is
known in the Iset basin (Ob basin). According to Paland-
aci¢ et al. (2017, 2020), mtDNA of this species (Clade 17)
was detected in Scandinavia and the British Isles, sug-
gesting that minnows from these areas also belong to the
species P. isetensis.

Dyldin et al. (2023: 671) pointed out the distributional
range of P. isetensis as “Arctic Ocean basin, from Murman
coast to East Siberian Sea basin (Kolyma basin); rivers of
northern and western Sea of Okhotsk basin (Ola and Uda
rivers); rivers of Peter the Great Bay drainage, probably
Amur River basin, and northwestern Sakhalin Island.”
This data only partially corresponds to the above-men-
tioned range. See further explanations in the discussion.

Phoxinus isetensis prefers rivers with fast-flowing wa-
ter that are rheophilic. In the northern regions, it also in-
habits riverbeds of large rivers, lakes, and brackish waters
(Berg 1949: 590; Tsvelev 2007: 277; our data).

Morphology comparison. PCA of 41 morphomet-
ric characters shows differences between P. isetensis
and P. adagumicus, P. chrysoprasius, and P. colchicus
from the Crimean Peninsula and the Caucasus (Fig. 7).
The greatest difference is from P. colchicus (no overlap),
while the remarkable overlap with the other two species
(P. adagumicus and P. chrysoprasius) is noted.

Compared to P. abanticus from the Lake Abant basin
in Turkiye (Turan et al. 2023), P. isetensis has scales on
the breasts in both sexes (vs. absence of scales on the
breast in males); 18—20 rays in the caudal fin (vs. 15—16
rays); and a more slender caudal peduncle (6.6—9.5, mean
8.0 vs. 11.0-12.7, mean 12.0).

Compared to P. adagumicus from the Kuban basin (Ar-
taev et al. 2024), P. isetensis has fewer scale rows above
the lateral line (10—21, mean 15.1, vs. 15—24, mean 18.4);
more total vertebrae (39-43, mean 41.0, mode 41, vs. 39—
42, mean 40.4, mode 40); more caudal vertebrae (16—21,

zse.pensoft.net

Artaev, O.N. et al.: Redescription of Phoxinus isetensis — the most widespread minnow in Europe

mean 18.9, mode 19, vs. 16-19, mean 18.0, mode 18);
double-row pharyngeal teeth with modal formula 2.5—4.2
(vs. single-row pharyngeal teeth with modal formula 5—4)
(Suppl. material 4).

Compared to P. bigerri from the Adour and Ebro ba-
sins in France and Spain (Kottelat 2007), P. isetensis has
fewer scale rows above the lateral line (10-21, mean
15.1, vs. 19-23).

Compared to P. chrysoprasius from the rivers of the
Crimean Peninsula (Artaev et al. 2024; Bogutskaya et al.
2023), P. isetensis has a slightly selender caudal pedun-
cle (minimum depth of caudal peduncle (6.6—9.0% SL,
mean 7.7 in females and 7.2—9.5, mean 8.2 in males (vs.
8.49.9, mean 9.1 in females and 8.1—-11, mean 9.9 in
males); slightly fewer circumpeduncular scales—28-45,
mean 35.3 (vs. 41-55, mean 46.2); more total number of
vertebrae—39—-43, mean 41.0, mode 41 (vs. 38-42, mean
40.4, mode 40); more number of caudal vertebrae—16—21,
mean 18.9, mode 19 (vs. 16-20, mean 18.0, mode 18)
(Suppl. material 4).

Compared to P. colchicus from the Black Sea coast
of the Caucasus and Kuban basin (Artaev et al. 2024;
Bogutskaya et al. 2023), P. isetensis in both sexes have
a Slenderer caudal peduncle (minimum depth of cau-
dal peduncle in percentage of caudal peduncle length
26.9-40.6, mean 32.5; vs. 42.0-58.6, mean 49.5); (Suppl.
material 3); less number of circumpeduncular scales —
28-45, mean 35.3 (vs. 36-48, mean 40.9): less number of
scales above lateral line — 10-21, mean 15.1 (vs. 16—23,
mean 18.8); fewer scale rows below lateral line — 8-16,
mean 11.2 (vs. 11-17, mean 13.4); different patterns of
scalation on the breast and anterior belly — 3 —6™ types,
mode 4" (vs. 3-10", 13" and 14", modal 6"); more to-
tal number of vertebrae — 39-43, mean 41.0, mode 41
(vs. 39-42, mean 40.1, mode 40); more number of caudal
vertebrae — 16—21, mean 18.9, mode 19 (vs. 16-19, mean
17.6, mode 18) (Suppl. material 4).

Compared to P. csikii from the Danube River basin,
Montenegro, and Bulgaria (Bogutskaya et al. 2019, 2023),
P. isetensis has a different pattern of scalation on the breast
and anterior belly—3"-6" types, mode 4" (vs. 3-9" types,
11", mode 7"); more total number of vertebrae—39-43,
mean 41.0, mode 41 (vs. 38-42, mean 40.1, mode 40);
more number of caudal vertebrae—16—21, mean 18.9, mode
19 (vs. 15-19, mean 17.4, mode 17); smaller difference be-
tween numbers of abdominal and caudal vertebrae—O—7,
mean 3.2 (vs. 2-9, mean 5.4) (Suppl. material 4).

Compared to P. krkae from the Krka River, Croatia
(Bogutskaya et al. 2019), P. isetensis has a greater number
of lateral-line scales (pored) 12-94, mean 53.7 (vs. 11-
54, mean 32.6); different pattern of scalation on the breast
and anterior belly—3"—6" types, mode 4" (vs. 3-7" types,
mode 6"); more total number of vertebrae—39-43, mean
41.0, mode 41 (vs. 37-40, mean 38.4, mode 39); more
number of caudal vertebrae—16—21, mean 18.9, mode 19
(vs. 15-18, mean 16.8, mode 17) (Suppl. material 4).

Compared to P. /umaireul Clades 1a and 1b from riv-
ers in Adriatic and Black Sea basins in Italy, Slovenia,
and Croatia (Bogutskaya et al. 2019), P. isetensis has a

Zoosyst. Evol. 100 (3) 2024, 1155-1173

PecPelv.SL

PreAn.SL a

1167

rePelv.SL

ie...
0.1 =» aA Aad APredors: /
AA r\ Ke A
A Predors,HL
A A a
BodyWid.SL
freies aM - & >
ero. y BodyDep.CaudPedDep!
a gy Ks é Aa> & Eye
Mary, MaxHeadwig H y rt .
MinDeptCaudPed.LénCaudPed Boy, Interorb. si : a
- 28 AAA &
“ A
| A
' SpoutLen.HL A « |i
90 Mi gy A ae Wide EN amen, A
x H EyeH i fii a
& Meaavepn ie oe en hs = - 1D . q d A
Io ir Interorb.EyeHorDia eno So. a} ga
~ nriq@ese: @ HeadLengl ja =
> HeadEop Nepal ostdB gi a ® aA ‘\ oR = CaudPedLen.CaudPedDert
oO SnoutLen.EyeHorDiam FinB sath raul mB,
= inBase.SL BP io; 5) Ab ,
m@ HeadDepNape.EyeHorDiam 2 | iat F a / |
. . :\/ @ m
. A nu
a | read el @Dayoen
ee PedL aL _
en.
wh ™ = 6h
-0.1 PecFinben.SL if a
|
Bt aFinDept.sL P. isetensis
PelFinLen.SL H P. adagumicus
>. PecFinLen.PecPelv P. chrysop FaSiUs

=

., /

-0.1

/

0.0
PC1 (29.23%)

[| P. colchicus

A Female
g Male

0.1

Figure 7. PCA based on 41 morphometric characters for Phoxinus spp. — P. isetensis, P. chrysoprasius from the Crimean Peninsula,

P. colchicus, and P. adagumicus from the Caucasus — and loading

plot showing how strongly each character influences principal

components. Samples of P. isetensis from the type locality (Severka River) are encircled by black.

different pattern of scalation on the breast and anterior
belly—3"6" types, mode 4" (vs. 2"-7" types, mode 3“);
more total number of vertebrae—39-43, mean 41.0, mode
41 (vs. 37-41, mean 39.3, mode 39); more number of
caudal vertebrae—16—21, mean 18.9, mode 19 (vs. 16—19,
mean 17.5, mode 18) (Suppl. material 4).

Compared to P. marsilii from the Danube River basin,
Austria, and Croatia (Bogutskaya et al. 2019, 2023), P. is-
etensis has a different pattern of scalation on the breast
and anterior belly—3—6" types, mode 4" (vs. 3™-8"
types, mode 6"); more total number of vertebrae—39-43,
mean 41.0, mode 41 (vs. 38-42, mean 40.1, mode 40)
(Suppl. material 4).

Compared to P. radeki from the Ergene River (Aegean
Sea basin) in Turkiye (Bay¢elebi et al. 2024), P. isetensis
has a higher number of scales above the lateral line-10-
21 (vs. 9-15) and below the lateral line—8—16 (vs. 6-9).

Compared to P. septimaniae from the Herault River,
France (Bogutskaya et al. 2019), P. isetensis has a dif-
ferent pattern of scalation on the breast and anterior bel-
ly—3"_6" types, mode 4" (vs. 12'-14" types, mode 14");
a greater total number of vertebrae—39—43, mean 41.0,

mode 41 (vs. 37-41, mean 39.3, mode 39); a greater num-
ber of caudal vertebrae—16—21, mean 18.9, mode 19 (vs.
16-19, 21, mean 17.6, mode 18) (Suppl. material 4).

Compared to P. strandjae from the rivers of the Black
Sea basin, Bulgaria, and the rivers of the Marmara Sea,
Turkiye (Bogutskaya et al. 2019, 2023), P. isetensis has
a different pattern of scalation on the breast and anteri-
or belly—3™—6" types, mode 4" (vs. 3-12" types, mode
11"); a greater total number of vertebrae—39—43, mean
41.0, mode 41 (vs. 38-42, mean 39.8, modal 40); a great-
er number of caudal vertebrae—16—21, mean 18.9, mode
19 (vs. 16-19, mean 17.7, mode 18) (Suppl. material 4).

Compared to P. strymonicus from the Strymon basin in
Greece and Macedonia (Kottelat, 2007), P. isetensis has
a smaller number of scales above the lateral line: 10-21
(vs. 21-24).

Compared to P. cf. ujmonensis from the Mundybash
River in Altai part of the Ob basin (Suppl. material 4),
P. isetensis has a smaller number of scales above the lat-
eral line: 10-21, mean 15.1 (vs. 15—19, mean 17.3); anda
number of scales below the lateral line: 8-16, mean 11.2
(vs. 12-15, mean 12.9).

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Discussion

Our study recognized the unnamed Phoxinus sp. with the
largest range in Europe (Clade 17 sensu Palandacic¢ et al.
2017) as P. isetensis, the species that was described from
the upper reach of the Chusovaya River (Kama- Volga
basin) and the Iset River basin (Ob basin) in the Middle
Urals. We redescribed the morphology and significantly
clarified the distributional range of this species. Accord-
ing to our results, P. isetensis can be distinguished from
other neighbor species by the large total number of verte-
brae — 39-43 (mode 41) (vs. 37-42), primarily due to an
increase in the number of caudal vertebrae — 16—21 (mode
19) (frequency distribution see in Suppl. material 4) and
in sequences of COI mtDNA (minimal p-distance = 0.05
to closely related P. colchicus). Remarkably, there is some
discrepancy between the genetic and morphological simi-
larities of P. isetensis. Being close genetically to P. colchi-
cus from the West Caucasus, it has more significant mor-
phological differences with this species compared to other
phylogenetically more distant species, P. adagumicus and
P. chrysoprasius (Figs 2, 7). In particular, these species
are more similar to P. isetensis in total number of verte-
brae and number of caudal vertebrae (Suppl. material 4).
Of the eighteen populations of P. isetensis, seventeen had
modes of 41 or 42 vertebrae, and only one population from
the Baltic Sea basin had 40 vertebrae. This main morpho-
logical difference — an increased number of vertebrae and
northernmost range relative to other European Phoxinus
— 1s consistent with Jordan’s rule (vertebral number in fish
increases with latitude) (McDowall 2008). However, it 1s
noteworthy that within the species range extended in the
latitudinal direction for more than 2000 km within 51-69
northern latitudes, there was no correlation.

Phoxinus isetensis redescribed in this study was re-
cently revalidated in Dyldin et al. (2023), but the authors
outlined its range mistakenly for the whole of West and
East Siberia as well as for Northeast Asia and the Far East
due to the absence of genetic and morphological data. Ac-
cording to our genetic results, the P. isetensis described
from the rivers near Yekaterinburg belonging to both the
upper stream of left tributaries of the Ob River and the
Chusovaya River itself (Kama-Volga basin) are identi-
cal or very similar (conspecific) to other samples from
the Volga basin, the Ural basin, and the remaining range
as outlined for Clade 17 sensu Palanda¢i¢ et al. (2017).
Therefore, P. isetensis has a huge range in northern and
eastern Europe, extending approximately 3.800 km from
west to east and 2.000 km from north to south. Its range in
Europe includes the basins of the Caspian, Baltic, White,
Barents, and North Seas. In the Caspian Sea basin, it is
widespread in the Upper and Middle Volga and Kama ba-
sins. In the Baltic Sea basin, it is widespread in the north-
ern and eastern parts. In the North Sea basin, it is noted for
Scandinavia and the British Isles (Palanda¢ic¢ et al. 2017).

To the best of our knowledge, the occurrence of P. is-
etensis in Asia is restricted to the Ural basin (Caspian Sea
basin) and the Iset basin (Tobol River — Irtysh River > Ob
River, which flows into the Kara Sea). The Ural Mountains

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Artaev, O.N. et al.: Redescription of Phoxinus isetensis — the most widespread minnow in Europe

are a well-known biogeographic barrier between Europe
and Asia (Abell et al. 2008), which, however, is character-
ized by the corridor linking Europe and Asia in the Middle
Ural via the Chusovaya River Valley. It was assumed this
corridor might facilitate aquatic fauna exchange between
Siberia (Ob basin) and European Russia (Volga basin) (Ko-
starev 1973; Makhrov et al. 2021). The Chusovaya River
belongs to the Kama- Volga basin, but its upper reaches are
located in Siberia — eastward the main Ural ridge and shar-
ing a watershed with the Ob basin through the Iset river ba-
sin. We have to note that in the upper reaches of the Chuso-
vaya River, there are a number of man-made reservoirs.
One of those, the Volchikha Reservoir, has a connection
through a small artificial canal to the Ob basin via the Re-
shotka River, a tributary of the Iset River. The reservoir and
canal are dated to the mid-1940s (Korlyakov and Nohrin
2014). Although it can be assumed that an artificial canal
could facilitate the dispersal of P. isetensis from the Ka-
ma-Volga system to the Ob basin, populations of minnow,
at least in the Iset basin, apparently colonized the Ob ba-
sin rather earlier. First, it is supported by early evidence of
the presence of conspecific populations in both the Ob and
Chusovaya basins by Lepechin (1772) and Georgi (1775).
This is further supported by the fact that not only the Sever-
ka (type locality), but other rivers in the Iset basin, inhab-
ited just one species of minnow, P. isetensis — for instance,
in the Kushtumga River (genetic data), which is located ca.
750 km along the riverbed from the canal connecting the
Volga and Ob basins near Yekaterinburg (Fig. 1).

Our finding in the distributional pattern of P. isetensis
is additional evidence of faunal exchange between Sibe-
ria and Europe via the Chusovaya River Valley. Contrary
to previous observations and suggestions on fish migra-
tions from Siberia to Europe (Kostarev 1973; Maric¢ et al.
2014; Perdices et al. 2015; Levin et al. 2017; Zinoviev
and Bogdanov 2017; Vinarski et al. 2021), the case of
P. isetensis suggests that fish migrations took place in the
reverse direction too. Remarkably, the eastward direction
of the recent (seemingly postglacial) colonization pattern
of P. isetensis is also corroborated by its presence in the
Upper and Middle Ural systems.

Noteworthy, one more Phoxinus species inhabits
the Ob basin — P. ujmonensis, described from the upper
tributaries of the Ob in the Altai Mountains. This species
is genetically distant from P. isetensis (Fig. 2, Suppl. ma-
terial 7). Our unpublished molecular data indicate that
the distribution of P. ujmonensis in the Ob basin is not
restricted by the Altai Mountains. The distribution of both
species in the Ob basin needs further clarification.

Based on the current distribution of P. isetensis (Fig. 1),
this species might rapidly colonize large northern areas
following the deglaciation of the last Pleistocene gla-
ciers. This is in line with the previously proposed hypoth-
esis about the colonization of Scandinavia by freshwater
fish from the southeast (Museth et al. 2007). Within the
boundaries of the Last Glacial Maximum (LGM), the
habitation of two other Phoxinus species (clades) is con-
firmed — P. cf. morella and Clade 10 (P. phoxinus) from
Ireland, with an unknown origin of population that might

Zoosyst. Evol. 100 (3) 2024, 1155-1173

be introduced — Denys et al. (2020) (Fig. 1). However,
northern populations of those species are located on the
southern periphery of the LGM, while P. isetensis inhab-
its most of the deglaciated region. One may suggest that
during the colonization of new habitats after LGM, P. is-
etensis could hybridize with other minnow species. This
hypothesis may be supported by a decrease in the total
number of vertebrae (mean 40.5 vs. 41.1 in the rest of the
range, Suppl. material 4) in the potential contact zone —
the east of the Baltic Sea basin—and it should be tested
in the future. It 1s noteworthy that in northern Norway,
the Phoxinus minnow has been a significant expansion
of the range as a result of introductions since the 1900s
(Museth et al. 2007); its ancient DNA was not found in
the sediments dating to 5.6—13 thousand years ago from
the Storsteinhola cave, while the ancient DNA of species
currently sharing biotopes with Phoxinus (Barbatula sp.
and Gobio gobio) was found (Boilard et al. 2024). Seem-
ingly, along with the post-glacial expansion of P. isetensis
northward, a shrinking of the range from the south can
be suggested. For example, Falk (1786: 432) recorded
Cyprinus phoxinus for the Volga and small rivers (“Zari-
za, Jelschanka. Sarpa u. f.”) near Volgograd city in Rus-
sia (Fig. 1). This is > 450 km southward from the most
southern contemporary records in the upper reaches of
the Tereshka (Artemyeva and Selishchev 2005) and Sura
basins within the Volga system. Although the right drain-
age of the Volga north of Volgograd 1s steep and rich in
small tributaries with fast-flowing water, minnows were
not recorded in this region. Probably populations in these
small rivers were extinct, being vulnerable to changes in
the environment, including general climate warming.

Acknowledgements

The authors are very grateful to taxonomist Boris Ka-
taev for his valuable advice on zoological taxonomy
and nomenclature. We are very thankful to Alexandra
Komarova, who helped with sampling. This study was
supported by the Russian Science Foundation, grant no.
24-44-20019, “Fishes of the Caspian Sea basin: genetic
diversity, evolution, and biogeography”.

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eZ

Supplementary material |
Additional and comparative materials

Authors: Oleg N. Artaev, Aleksey A. Bolotovskiy, Ilya
S. Turbanov, Alexander A. Gandlin, Aleksey V. Kutu-
zov, Marina A. Levina, Danila A. Melentev, Ivan V.
Pozdeev, Mikhail Ya. Borisov, Boris A. Levin

Data type: docx

Explanation note: Additional material on Phoxinus is-
etensis and comparative material on P. adagumicus,
P. chrysoprasius and P. colchicus.

Copyright notice: This dataset is made available under
the Open Database License (http://opendatacommons.
org/licenses/odbl/1.0/). The Open Database License
(ODbL) is a license agreement intended to allow us-
ers to freely share, modify, and use this Dataset while
maintaining this same freedom for others, provided
that the original source and author(s) are credited.

Link: https://do1.org/10.3897/zse.100.126702.suppl1

Supplementary material 2

Primary morphological data from type
locality

Authors: Oleg N. Artaev, Aleksey A. Bolotovskiy, Ilya
S. Turbanov, Alexander A. Gandlin, Aleksey V. Kutu-
zov, Marina A. Levina, Danila A. Melentev, Ivan V.
Pozdeev, Mikhail Ya. Borisov, Boris A. Levin

Data type: xlsx

Explanation note: Primary morphological data of Phoxi-
nus isetensis from type locality (Severka River).

Copyright notice: This dataset is made available under
the Open Database License (http://opendatacommons.
org/licenses/odbl/1.0/). The Open Database License
(ODbL) is a license agreement intended to allow us-
ers to freely share, modify, and use this Dataset while
maintaining this same freedom for others, provided
that the original source and author(s) are credited.

Link: https://do1.org/10.3897/zse.100.126702.suppl2

zse.pensoft.net

Artaev, O.N. et al.: Redescription of Phoxinus isetensis — the most widespread minnow in Europe

Supplementary material 3
Comparsion of morphometrics

Authors: Oleg N. Artaev, Aleksey A. Bolotovskiy, Ilya
S. Turbanov, Alexander A. Gandlin, Aleksey V. Kutu-
zov, Marina A. Levina, Danila A. Melentev, Ivan V.
Pozdeev, Mikhail Ya. Borisov, Boris A. Levin

Data type: xlsx

Explanation note: Morphometrics of Phoxinus isetensis,
P. adagumicus, P. chrysoprasius, P. colchicus and its
comparison.

Copyright notice: This dataset is made available under
the Open Database License (http://opendatacommons.
org/licenses/odbl/1.0/). The Open Database License
(ODbL) is a license agreement intended to allow us-
ers to freely share, modify, and use this Dataset while
maintaining this same freedom for others, provided
that the original source and author(s) are credited.

Link: https://doi.org/10.3897/zse.100.126702.suppl3

Supplementary material 4

Meristic and qualitative characters

Authors: Oleg N. Artaev, Aleksey A. Bolotovskiy, Ilya
S. Turbanov, Alexander A. Gandlin, Aleksey V. Kutu-
zov, Marina A. Levina, Danila A. Melentev, Ivan V.
Pozdeev, Mikhail Ya. Borisov, Boris A. Levin

Data type: xlsx

Explanation note: Meristic and qualitative characters of
Phoxinus isetensis and other Phoxinus species pub-
lished in the literature.

Copyright notice: This dataset is made available under
the Open Database License (http://opendatacommons.
org/licenses/odbl/1.0/). The Open Database License
(ODbL) is a license agreement intended to allow us-
ers to freely share, modify, and use this Dataset while
maintaining this same freedom for others, provided
that the original source and author(s) are credited.

Link: https://doi.org/10.3897/zse.100.126702.suppl4

Zoosyst. Evol. 100 (3) 2024, 1155-1173

Supplementary material 5
Material for genetic studies

Authors: Oleg N. Artaev, Aleksey A. Bolotovskiy, Ilya
S. Turbanov, Alexander A. Gandlin, Aleksey V. Kutu-
zov, Marina A. Levina, Danila A. Melentev, Ivan V.
Pozdeev, Mikhail Ya. Borisov, Boris A. Levin

Data type: xlsx

Copyright notice: This dataset is made available under
the Open Database License (http://opendatacommons.
org/licenses/odbl/1.0/). The Open Database License
(ODbL) is a license agreement intended to allow us-
ers to freely share, modify, and use this Dataset while
maintaining this same freedom for others, provided
that the original source and author(s) are credited.

Link: https://do1.org/10.3897/zse.100.126702.suppl5

Supplementary material 6
Best partition schemes

Authors: Oleg N. Artaev, Aleksey A. Bolotovskiy, Ilya
S. Turbanov, Alexander A. Gandlin, Aleksey V. Kutu-
zov, Marina A. Levina, Danila A. Melentev, Ivan V.
Pozdeev, Mikhail Ya. Borisov, Boris A. Levin

Data type: docx

Explanation note: The best partition schemes generat-
ed by ModelFinder v.2.2.0 (ML) and PartitionFinder
v2 1 -€BIy

Copyright notice: This dataset is made available under
the Open Database License (http://opendatacommons.
org/licenses/odbl/1.0/). The Open Database License
(ODbL) is a license agreement intended to allow us-
ers to freely share, modify, and use this Dataset while
maintaining this same freedom for others, provided
that the original source and author(s) are credited.

Link: https://do1.org/10.3897/zse. 100. 126702.suppl6

1173

Supplementary material 7
ML tree

Authors: Oleg N. Artaev, Aleksey A. Bolotovskiy, Ilya
S. Turbanov, Alexander A. Gandlin, Aleksey V. Kutu-
zov, Marina A. Levina, Danila A. Melentev, Ivan V.
Pozdeev, Mikhail Ya. Borisov, Boris A. Levin

Data type: docx

Explanation note: ML phylogenetic tree of concatenated
COI and cytb mtDNA sequences.

Copyright notice: This dataset is made available under
the Open Database License (http://opendatacommons.
org/licenses/odbl/1.0/). The Open Database License
(ODbL) is a license agreement intended to allow us-
ers to freely share, modify, and use this Dataset while
maintaining this same freedom for others, provided
that the original source and author(s) are credited.

Link: https://do1.org/10.3897/zse.100.126702.suppl7

Supplementary material 8
Genetic p-distances

Authors: Oleg N. Artaev, Aleksey A. Bolotovskiy, Ilya
S. Turbanov, Alexander A. Gandlin, Aleksey V. Kutu-
zov, Marina A. Levina, Danila A. Melentev, Ivan V.
Pozdeev, Mikhail Ya. Borisov, Boris A. Levin

Data type: xls

Explanation note: estimates of average evolutionary di-
vergence over sequence pairs within and between
Phoxinus species and clades.

Copyright notice: This dataset is made available under
the Open Database License (http://opendatacommons.
org/licenses/odbl/1.0/). The Open Database License
(ODbL) is a license agreement intended to allow us-
ers to freely share, modify, and use this Dataset while
maintaining this same freedom for others, provided
that the original source and author(s) are credited.

Link: https://do1.org/10.3897/zse.100.126702.suppl8

zse.pensoft.net